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PhD Defense, Abhinav Sharma, " MICRO-PHYSIOLOGICAL MODELS TO MIMIC MUCOSAL BARRIER COMPLEXITY OF THE HUMAN INTESTINE IN VITRO"

Date/Time: 

Tuesday, March 31, 2020 - 1:00pm

Location: 

Via Zoom

Details: 

Jungwoo Lee, Co-Chair, Chemical Engineering
Neil Forbes, Co-Chair, Chemical Engineering

ABSTRACT

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while 
protecting internal  tissues  and  maintaining  symbiotic  relationship  with  luminal  microbiota. 
 This  bio-barrier  consists  of  a cellular  epithelial  barrier  and  an  acellular  mucus  
barrier.  Secreted  mucus  regulates  barrier  function  via  in  situ biochemical  and  
biophysical  interaction  with  luminal  content  that  continually  evolves  during  digestion  
and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain 
dynamic homeostasis of the  gastrointestinal  tract.  However,  the  importance  of  mucus  barrier 
 has  been  largely  underrated  for  in  vitro mucosal  tissue  modeling.  The  major  gap  is  
the  lack  of  experimental  material  (i.e.  functional  mucins)  and platforms to integrate a 
relevant thickness of mucus layer with an epithelium under physiological conditions.


Here we report our progress on developing human-relevant micro-physiological models of the mucosal 
barrier in static and dynamic settings by using natural mucins derived from a porcine small 
intestine (PSI). To overcome limited availability of functional mucus, we first developed a simple 
and scalable protocol for natural mucus extraction by directly solubilizing a relatively sterile 
inner mucus layer from PSI that is readily accessible. Subsequently, functional separation of mucin 
proteins was performed by exploiting pH-dependent reversible sol- gel  transition.  Under optimized 
 alkaline  condition  (0.01M  NaOH),  the  mucus  layer was  selectively  solubilized from the 
mucosal surface with a 72% yield (1275 mg/m PSI). The extracted and purified natural mucins 
retained essential  biophysical  and  biochemical  characteristics.  The  in  vitro  mucus  barrier 
 model  enabled  us  to  discover ionic (Ca2+) environment dependent mucus barrier and its 
transport properties.


The mucus barrier was successfully integrated with human epithelial cell layer (HT-29), which 
allowed the studies of bi-directional crosstalk between luminal content and tissue immune cells 
through a physiologically relevant  mucosal  interface.  The  applied  mucus  barrier  did  not  
cause  any  cytotoxic  or  immunogenic  effects  to human intestinal and immune cells. As expected, 
mucus prevents the transmigration of probiotic bacteria VSL#3. In  the  absence  of  mucus,  these  
bacteria  caused  epithelial  damage,  immune  cell  differentiation  and  induced production of 
pro-inflammatory cytokines IL-8 and TNF-α. The most intriguing result from these studies was that 
mucus increased the transmigration of pathogenic Salmonella. Similar to the transmigration of 
probiotic bacteria, breach of the mucosal barrier by Salmonella induced production of IL-8 and 
TNF-α. The importance of bacterial motility  was  confirmed  by  showing  that  Salmonella  with  a 
 knockout  that  prevents  flagella  formation  does  not penetrate  the  barrier.  Co-cultures  of 
 VSL#3  and  Salmonella  in  the  mucosal  barrier  platform  demonstrated  the differences in 
epithelial and immune cell responses under symbiosis or dysbiosis like conditions.


Taking bioengineering approaches, we have developed mucosal barrier models of intestines. 
Established models represent cellular and extracellular complexities in a controlled and accessible 
manner. We envision that in vitro  mucosal  barrier  models  will  serve  as  an  enabling  tool  
for  understanding  basic  biology  and  disease
progression in the intestines.

 

 
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